The proposal is to investigate dynamic and static processes in the interaction between steroids and steroid-binding proteins. The purpose is to develop a better understanding of how the steroids are bound and the basis for their specificity. By taking advantage of known photophysical properties of extrinsic and intrinsic luminescent probes, using steady state and nanosecond time-resolved fluorescence spectroscopy, a more detailed picture will be developed about the steroid binding site and its relationship to protein function and structure. The work will focus on two serum proteins, sex steroid-binding protein (SBP) and corticosteroid-binding globulin (CBG), using species comparison to help elucidate the basis for steroid specificity. The long range goal is to find out how steroids interact with intra-cellular receptor proteins. The immediate goal is to understand the serum proteins. The methods to be used in these studies will be time-resolved and steady state fluorescence and steroid binding assays. The specific aims are: 1. The two-state fluorescence decay kinetics of equilenin bound to human and rabbit SBP will be studied to provide information about the chemical environment of the steroid binding site and interactions with specific amino acid residues. 2. Time-resolved fluorescence anisotropy decay measurements of probes bound to SBP and CBG will be carried out to obtain information about the hydrodynamic shape and structural stability of the two proteins. 3. Steroid-like fluorescent probes will be studied which potentially have binding affinity for SBP and CBG, including carcinogenic aromatic hydrocarons reported to have mild estrogenic activity. 4. The interaction of Ca++ with SBP will be studied using lanthanides. 5. The role of the protein carbohydrate will be investigated to learn how it affects the structural integrity and steroid binding of SBP and CBG.